LOCUS       sequence-341668-        4950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector expressing scrambled shRNA and a nuclear EYFP 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4950)
  AUTHORS   Venniro M, Russell TI, Ramsey LA, Richie CT, Lesscher HMB, 
            Giovanetti SM, Messing RO, Shaham Y
  TITLE     Abstinence-dependent dissociable central amygdala microcircuits 
            control drug craving.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Apr 7;117(14):8126-8134. doi: 
            10.1073/pnas.2001615117. Epub 2020 Mar 23.
  PUBMED    32205443
REFERENCE   2  (bases 1 to 4950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.2001615117"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2020-04-7- 7"; volume: "117"; issue: "14"; pages: "8126-8134"
COMMENT     Sequence Label: pOTTC1535	pscAAV mU6 shRNA(scram) CMV-IE Nuc-EYFP
FEATURES             Location/Qualifiers
     source          1..4950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      52..507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(139..158)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     349..370
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(524..543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     643..665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(703..721)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        789..893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(894..962,963..1754)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1112..1131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1925..2513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2414..2433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        complement(2761..3073)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(2822..2842)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3168..3471
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3472..3675
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3626..3646
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(3735..3737,3738..3740,3741..4451)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3780..3801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4041..4060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4388..4409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4476..4496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4500..4520
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4558..4575)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4564..4675
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4787..4927
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4787..4916
                     /label=AAV2 ITR
ORIGIN
        1 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
       61 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      121 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      181 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      241 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
      301 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      361 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
      421 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
      481 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
      541 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      601 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      661 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
      721 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
      781 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      841 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      901 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      961 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1021 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1081 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1141 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1201 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1261 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1321 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1381 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1441 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1501 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1561 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1621 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1681 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1741 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1801 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1861 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1921 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1981 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2041 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     2101 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2161 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2221 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2281 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2341 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2401 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2461 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2521 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca
     2581 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc
     2641 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ctgcggccgc acgcgtcgac
     2701 ctcgagaaaa aagcgcttag ctgtaggatt ctctcttgaa gaatcctaca gctaagcgca
     2761 aacaaggctt ttctccaagg gatatttata gtctcaaaac acacaattac tttacagtta
     2821 gggtgagttt ccttttgtgc tgttttttaa aataataatt tagtatttgt atctcttata
     2881 gaaatccaag cctatcatgt aaaatgtagc tagtattaaa aagaacagat tatctgtctt
     2941 ttatcgcaca ttaagcctct atagttacta ggaaatatta tatgcaaatt aaccggggca
     3001 ggggagtagc cgagcttctc ccacaagtct gtgcgagggg gccggcgcgg gcctagagat
     3061 ggcgcgtcgg atctctagag cgggcccggt aacgcgtgga tctaatatag ttattaatag
     3121 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     3181 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     3241 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     3301 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     3361 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     3421 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     3481 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     3541 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     3601 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     3661 tatataagca gagctggttt agtgaaccgt cagatccgaa gttatcgcta gcgctaccgg
     3721 actcagatct caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3781 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     3841 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     3901 tgccctggcc caccctcgtg accaccttcg gctacggcct gcagtgcttc gcccgctacc
     3961 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4021 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4081 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4141 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4201 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     4261 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4321 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc
     4381 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4441 agctgtacaa gtccggcctg cgcagccgcg ccgacccaaa aaagaagcgc aaggtagacc
     4501 ccaagaagaa gcgcaaggta gatccaaaaa agaagagaag gtatgatagt ctagaagcct
     4561 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4621 ccctggaagg tgccactccc accgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4681 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4741 attgggaaga caatagcagg catgctgggg cggaccgagc ggccgcagga acccctagtg
     4801 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4861 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4921 ctgcaggggc gcctgatgcg gtattttctc
//
